Effects of Dense Granular Protein 6 (GRA6) Disruption on Neospora caninum Virulence

Abstract

Neospora caninum (N. caninum) is a major cause of abortions in cattle. During its invasion of host cells, a parasitophorous vacuole (PV) is formed, accompanied by an intravacuolar network (IVN). The IVN takes part in parasite ingesting of nutrients from hosts. The dense granular proteins of N. caninum (NcGRAs) play a key role in forming the PV and the IVN, which may influence virulence during N. caninum invasion. The present study aimed to explore the biological function of NcGRA6 in N. caninum by disrupting the NcGRA6 gene in the Nc-1 strain. We successfully constructed an NcGRA6-targeting CRISPR plasmid (pNc-SAG1-Cas9:U6-SgGRA6) and amplified the DHFR-TS DNA donor. The NcGRA6 knockout mutation (NcGRA6) was generated by co-electroporation of the pNc-SAG1::CAS9-U6::sgGRA6 plasmid and the DHFR-TS DNA donor into the Nc-1 strain, which was then cultured under pyrimethamine selection pressure. The NcGRA6 mutation was further verified by identification of NcGRA6 gene disruption using PCR, measurement of NcGRA6 gene transcription levels using qPCR, assessment of NcGRA6 protein expression levels using western blotting, and observation of NcGRA6 protein location using immunofluorescence and immunoelectron microscopy. The results of in vitro virulence assays, including plaque, invasion, egress, and replication assays, showed that the NcGRA6 strain had smaller plaques, similar invasion and egress ability, and slower intracellular replication ability than the Nc-1 strain. The results of in vivo virulence assays showed that the NcGRA6 strain exhibited reduced virulence and improved survival ability in mice compared with the Nc-1 strain. The parasite burden in NcGRA6 strain-infected mouse tissues, including the heart, brain, liver, spleen, lung, and kidney, was significantly reduced compared with that in mice infected with the Nc-1 strain. These data suggest that we successfully constructed a NcGRA6 strain and verify that NcGRA6 is a critical virulence factor. NcGRA6 gene disruption can slow down N. caninum proliferation and lower the pathogenicity to hosts. Our findings provide a foundation for future research on other targeted N. caninum protein functions and may help in exploring the interaction mechanisms between parasites and hosts.

Improvement to Gene Editing in Neospora Caninum: Knockout ku80 to Increase the Efficiency of Homologous Recombination

“…Some methods have been reported to recover the defect, including substituting the N. caninum speci c promoter and terminator [5,15]. In this study, deletion of ku80 was proved to work well.…”

“…The e ciency of CRISPR/Cas9 technology has always been an obstacle [15]. Several parameters affect the e ciency of gene targeting in CRISPR/Cas9 technology, including the tness score of the target gene, drug selectable markers, plasmid, the targeting DNA ank lengths, the life force of parasite and so on.…”

Disruption of Dense Granular Protein 2 (GRA2) Decreases the Virulence of Neospora caninum

“…To rapidly obtain the Nc-1 strain of N. caninum tachyzoites, tachyzoites were grown in fast-growing MDBK cells and purified as described previously (29). In brief, N. caninum tachyzoites were collected when 80% of the host cells were lysed and passed through a 27-gauge needle.…”

“…Similar to T. gondii, N. caninum tachyzoites replicate in the PV after invasion into host cells and then egress out and enter uninfected cells (35,36). The egress assays were performed as previously described (29). A total of 1 × 10 5 tachyzoites were inoculated onto HFF cells coated on coverslips in six-well plates under conditions of 37 • C and 5% CO 2 for 1 h, and then the extracellular parasites were discarded by washing three times with sterilized PBS.…”

“…The parasite burden was determined by measuring the Nc5 gene contents using an absolute qPCR assay with tissue genomic DNA as a template (37). The histopathological changes in the isolated tissues were determined by hematoxylin-eosin (HE) assay as previously described (29). Briefly, fresh tissues were washed with 0.9% NaCl, cut into patches, and immediately fixed in 4% paraformaldehyde.…”